1. Field of the Invention
This invention relates to activated lymphocytes derived from cord blood, pharmaceutical preparations containing the aforesaid activated lymphocytes as a main ingredient, and a method and kit for producing the aforesaid preparations for the purpose of treating various tumors and infection diseases and preventing these diseases from developing and redeveloping and hastening take of stem cells of various organs and so on.
2. Description of the Prior Art
Lately, intense interest has been shown toward lymphocytes bearing an immune system for biophylaxis. Specifically, T-lymphocytes are one of the important cells having an efficient cellular immunity and sorted into the following groups in accordance with the reactivity of monoclonal antibodies. For example, the T-lymphocytes having reactivity with anti-CD3 antibodies (xe2x80x9cCDxe2x80x9d is short for xe2x80x9ccluster of differentiation) belong to CD3 positive cells. There have been performed a number of studies of the relationship between the antigen manifested from these lymphocytes and their functions.
The lymphocyte manifesting CD45 RA-antigen among the CD3 positive cells is a naive T-lymphocyte, which is deemed not to have antitumor activity. On the contrary, the lymphocyte manifesting CD45 RO-antigen among the aforesaid CD3 positive cells is a memory T-lymphocyte, which is recognized to have the function of antitumor activity. Sekine, one of the inventors, has already reported that it is possible to proliferate the lymphocytes with a solidus anti-CD3 antibody and interleukin 2, so that autologous lymphocytes obtained as the result of proliferation can possess an antitumor function (Japanese Patent Public Disclosure HEI 03-80076(A)).
There have been made any reports that the lymphocytes derived from peripheral blood or the like can be proliferated by using the anti-CD3 antibody or interleukin 2, thus to produce autologous lymphocytes having an antitumor function. Further, Ito et al., some of the inventors of this invention, have reported that the autologous lymphocytes proliferated with the anti-CD3 antibody and interleukin 2 is effective against viral infections of a patient of innate immunodeficiency (Kimiya Ito and Teruaki Sekine, Monthly Magazine xe2x80x9cIgakunoayumixe2x80x9d Ishiyaku Pub. Inc., Vol. 181(1997), No. 6, pp. 426-427).
Bone marrow transplant is generally performed in a case that a patient and a donor are compatible with each other in leukocyte blood group (hereinafter abbreviated as xe2x80x9cHLAxe2x80x9d). However, since there are many types of HLA, it is remarkably rare that the HLAs of the patient and donor are identical with each other. With the existing state of affairs, the bone marrow transplant has been performed when only the chief components of the HLA are identical between the patient and donor. Disadvantageously, incomplete match of the HLA between the patient and donor possibly cause host diseases (hereinafter abbreviated as xe2x80x9cGVHDxe2x80x9d) in the bone marrow transplant.
For the purpose of treating the GVHD disease, which brings about a serious case, an immune inhibitor is used. The patient dosed with the immune inhibitor gets over the disease for the time being, but mostly gets into a serious situation of developing cytomegalovirus or Epstein-Barr virus infections, resulting in death. Thus, Elizabeth et al. reported that the cytomegalovirus infections of the patient of bone marrow transplant can be treated by inducing specific CD4 positive cells from the lymphocytes of the bone marrow donor, thereby to prevent and treat the virus infections caused in the immunosuppressive conditions (Elizabeth A. Walter, M.D. et al., The New England Journal of Medicine, Vol.333, pp.1038-1044).
Furthermore, allogenic lymphocytes prepared by a pheresis procedure have been clinically used for treating a leukemia patient, i.e. donor leukocyte transfusion (hereinafter abbreviated as xe2x80x9cDLTxe2x80x9d). Although DLT has a high therapeutic value, it was confirmed that the treatment using DLT caused 50% to 80% of the leukemia patients to suffer from acute GVHD and some 20% of the leukemia patients to suffer from lathal GVHD (Kolb. H. J. et al. Blood, Vol.86, pp.2041-2050 (1995), and Slavin S. et al., Experimental Hematology, Vol.23, pp.1553-1562 (1995)). It takes a long time to perform the pheresis, thus to impose an oppressive burden on the donor.
Giralt et al. reported about DLT using leukocyte free from CD8 positive cells (Giralt, S. et al., Blood, Vol.86, pp.4338-4343 (1995)). Ritz et al. reported about DLT using CD4 positive cells prepared by pheresis (Claret E J. et al., Journal of Clinical Investigation, Vol.100, No.4, pp.855-866 (1997)). It was reported that, in either case, the effect of GVL (abbreviated from xe2x80x9cGraft versus leukemiaxe2x80x9d), in which slight GVL favorably affects a patient, is created, but the GVL effect is weak.
Recently, there has been studied an attempt to use cells contained in cord blood instead of the cells prepared from peripheral blood or marrow blood, which has been practically superseded by a marrow-bone transplant. However, in general, the lymphocyte contained in the cord blood is a naive lymphocyte, which is not irritated by antigens, and thus, is usually deemed not to possess antitumor activity nor an antiviral effect.
The cord blood contained in an umbilical cord can be obtained without imparting a burden on the donor and is little in restriction on HLA. Therefore, it is advantageous to the donor in comparison with collection of cells from the bone marrow or peripheral blood. However, cord blood inoculation may possibly redevelop a tumor with greater frequency and is disadvantageous from the standpoint of the possibility of viral infections and the take of the cells inoculated.
An object of the present invention is to provide activated lymphocytes and pharmaceutical preparations containing the activated lymphocytes, which can be inoculated onto a receptor with secure against redevelopment of a tumor, eliminating the possibility of causing viral infections and recurring a tumor caused by cord blood transplant.
Another object of the present invention is to provide activated lymphocytes and pharmaceutical preparations prepared from activated lymphocytes, which has high ability of take of cells in the preparations inoculated.
Still another object of the present invention is to provide a method and kit, which can produce pharmaceutical preparations capable of being inoculated onto a receptor with secure against redevelopment of a tumor, eliminating the possibility of causing viral infections and recurring a tumor caused by cord blood transplant
To attain the objects described above according to the present invention, there is provided activated lymphocytes, which are derived from cord blood and proliferated. The lymphocytes derived from the cord blood is prepared by segregating lymphocytes from the cord blood and proliferating the segregated lymphocytes directly in vitro or segregating monocytes from cord blood and proliferating the monocytes in vitro. The segregated lymphocytes or monocytes are activated and proliferated with interleukin 2 and/or anti-CD3 antibody.
The lymphocytes derived from the cord blood according to the invention can be inoculated onto a receptor or a patient with secure against redevelopment of a tumor, eliminating the possibility of causing virus-infected diseases, i.e. viral infections, and recurring a tumor caused by cord blood transplant.
Pharmaceutical preparations according to the present invention are prepared by segregating monocytes from cord blood, activating and proliferating the monocytes thus segregated with interleukin 2 and/or anti-CD3 antibody to obtain activated and proliferated lymphocytes, and adding the activated and proliferated lymphocytes thereto as a main ingredient.
The pharmaceutical preparations according to the invention serves to treat a tumor and infection diseases and prevent these diseases from developing and redeveloping.
Further, the present invention provides a method and kit for producing activated lymphocytes derived from cord blood, comprising the processes of segregating monocytes from cord blood, and activating and proliferating the monocytes thus segregated with interleukin 2 and/or anti-CD3 antibody to obtain activated and proliferated lymphocytes.
According to the method and kit of the present invention, the desired high-ability activated lymphocytes and pharmaceutical preparations, which can be inoculated onto a receptor with secure against redevelopment of a tumor, eliminating the possibility of causing viral infections and recurring a tumor caused by cord blood transplant, can be obtained.
Other and further objects of this invention will become obvious upon an understanding of the embodiments described hereinafter or will be indicated in the appended claims, and various advantages not referred to herein will occur to one skilled in the art upon employment of the invention in practice.